Coronavirus 2′-O-methyltransferase: A promising therapeutic target

Highlights • Coronavirus NSP16 plays a critical role in viral RNA capping and evasion of innate immunity.• Coronavirus mutants lacking NSP16 activity are attenuated both in vitro and in vivo.• Attenuation of NSP16 mutants driven by activity of IFIT family members targeting viral RNA lacking a 2′O methylation on its cap.• Targeting NSP16 activity offers a broad therapeutic approach for treatment of current and future coronavirus outbreaks.


Introduction
Prior to the 21st century, coronaviruses (CoVs) were considered pathogens of minor concern to human health, known to cause cold-like symptoms and infrequently associated with more severe respiratory disease (Riski and Hovi, 1980).The current perception of CoVs is now much different.In 2019, severe acute respiratory syndrome CoV 2 (SARS-CoV-2), the causative agent of COVID-19, emerged in Wuhan, China under conditions that still remain unclear (Wang et al., 2020).Initially termed the Novel Coronavirus (nCoV)− 2019, the virus bore strong genetic resemblance to circulating viruses of the Sarbecovirus subgenus, or SARS-related CoVs, found in bats (Zhou et al., 2020).The prototypical SARS-CoV had previously emerged in 2002 in China (Low and McGeer, 2003).While more phylogenetically distant from the SARS-related CoVs, Middle East respiratory syndrome (MERS)-CoV is also capable of causing severe respiratory distress (Lamers and Haagmans, 2022), and continues to circulate at low frequencies in the Middle East (World Health Organization, 2022).Thus, the emergence of three highly pathogenic human CoVs since the start of the 21st century, combined with the presence of circulating CoVs in natural reservoirs, such as bats, underscores the persistent threat of future CoV emergence, the need to better understand CoV biology, and the potential benefits of developing better treatment strategies.

CoV replication and the nonstructural proteins
CoVs are positive-sense, non-segmented RNA viruses, which possess large RNA genomes, up to 31.7 kgbases (kb) (Woo et al., 2010).The viruses belong to the order Nidovirales (nidoviruses), and are so named because of the 3′ co-terminal, or nested (Latin: "nido"), set of sub-genomic RNA species that are generated during transcription of the viral genome.In addition to CoVs, nidoviruses include the newly distinguished Tobaniviridae family (which include the toroviruses), the genetically smaller viruses of the Arteriviridae family (arteriviruses), invertebrate viruses of the Roniviridae and Mesoniviridae families, and novel families recently delineated by the International Committee on Taxonomy of Viruses (https://ictv.global/taxonomy)(Gulyaeva and Gorbalenya, 2021;Snijder et al., 2016).
As positive-sense RNA viruses, nidoviruses, including CoVs, initiate translation of the viral genome upon release into the cytoplasm (V'kovski et al., 2021).Importantly, a large ORF at the 5′ end of the genome, comprising about two-thirds of the genome, ORF1ab, encodes a replication-transcription complex (RTC) which comprises, in CoVs, sixteen distinct nonstructural proteins (NSPs).The RTC localizes to virus-induced, endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs), which serve as virus replication factories; the DMVs also likely shield viral RNA from host pattern recognition receptors (PRRs) that detect viral RNA (Horova et al., 2021;Klein et al., 2020;Snijder et al., 2020;V'kovski et al., 2021).The NSPs perform such essential functions as replicating and transcribing the genome, capping the viral RNA, and proteolytically processing the ORF1ab polyprotein into the individual NSPs (Romano et al., 2020).
Nidoviruses possess many shared features of replication, including a polycistronic genome structure, the aforementioned ORF1ab RTC polyprotein that consists of two slightly overlapping ORFs (ORF1a and ORF1b), an encoded ribosomal frameshift mechanism that permits the extension of the second of these two ORFs from the first, a conserved nucleotidyltransferase domain adjacent to the RNA-dependent RNA polymerase (RdRp), and a conserved helicase-containing domain (Lehmann et al., 2015).Notably, CoVs possess NSP functions that are lacking in other nidoviruses (Snijder et al., 2016).Compared to the smaller genomes of arteriviruses, CoV genomes encode additional functional components including an RNA exonuclease (ExoN, NSP14), important for proofreading during replication (Denison et al., 2011), a guanine-N7 methyltransferase (N7-MTase, encoded on a separate domain of NSP14), and a 2′-O-MTase (NSP16), the latter two important in viral RNA capping (Lauber et al., 2013).
In addition to the KDKE motif, NSP16 MTase function depends on binding NSP10.Providing structural stability for both NSP16 and NSP14 (Krafcikova et al., 2020), NSP10 is required for NSP16 capacity to bind both RNA substrate and the methyl donor S-adenosyl-L-methionine (SAM) (Chen et al., 2011).Like NSP16, NSP10 is also well-conserved among CoVs (Abbasian et al., 2023;Li et al., 2023a).In the absence of NSP10, NSP16 MTase function is completely abrogated in biochemical studies (Bouvet et al., 2010).Targeting small peptides to the NSP10-NSP16 interface to prevent binding demonstrated that such peptides reduce NSP16 MTase activity and CoV replication in vitro.In addition, targeting the NSP10-NSP16 interface even protected mice from a uniformly lethal dose of 5 × 10 5 plaque-forming units (PFU) MHV-A59 administered intrahepatically, compared to a control peptide with scrambled sequence (Wang et al., 2015).Together, these results argue that both the KDKE motif as well as the interaction with NSP10 are necessary for CoV MTase activity.(Ivanov and Ziebuhr, 2004) followed by NSP12-mediated guanosine cap transfer (Walker et al., 2021).Alternatively, NSP9 may form an intermediate with the viral RNA itself prior to guanosine cap transfer (Park et al., 2022).Regardless, NSP14 methylates the N7 of the guanine base of the cap, resulting in cap0 RNA (Chen et al., 2009).Finally, NSP16 methylates the 2′-O of the ribose of the first transcribed nucleotide, resulting in cap1 RNA (Decroly et al., 2008).
Consistent with the data in vitro, CoV NSP16 mutants caused less severe disease in vivo, varying across different animal models (Menachery et al., 2017(Menachery et al., , 2014b;;Schindewolf et al., 2023;Ye et al., 2022;Züst et al., 2011).For MHV, the NSP16 mutant (NSP16-D130A) was highly attenuated after intraperitoneal injection with no detectable titer in either the liver or spleen (Züst et al., 2011); notably, a mutant lacking capacity to bind cap0 RNA (NSP16-Y15A) was similarly attenuated.Both BALB/c and C57BL/6 mice infected with the SARS-CoV NSP16-D130A mutant also displayed a sharp reduction in viral titer at later timepoints (4-and 7-days post-infection, DPI), reduced weight loss, and less diseased lung function than control mice (Menachery et al., 2014b).Moreover, similar gene expression in the lungs was observed at 1 and 2 DPI; decreases in immunity-related genes were observed in mutant-infected mice at 4 and 7 DPI, consistent with viral clearance (Menachery et al., 2018).For MERS-CoV, the NSP16-D130A mutant was attenuated (Menachery et al., 2017) in two different murine models (Cockrell et al., 2016;Zhao et al., 2014), showing a >2 log 10 -lower viral titer in the lung at 4 DPI, compared to wild-type (WT)-infected mice.Finally, SARS-CoV-2 NSP16-D130A mutants were attenuated in both a hamster model (Schindewolf et al., 2023;Ye et al., 2022) and a transgenic human ACE2-expressing mouse model (McCray et al., 2007;Ye et al., 2022).In each model, SARS-CoV-2 2′-O-MTase mutants had reduced viral load in the lung at 4 DPI and less lung disease than control animals.While immune responses varied across studies in the hamster model, viral attenuation was observed most strongly in the upper versus lower airway (Schindewolf et al., 2023;Ye et al., 2022), suggesting differential immune responses between tissues.Together, studies with NSP16 2′-O-MTase mutants demonstrate an essential function to CoVs which is necessary for infection and pathogenesis.

IFIT proteins restrict CoV NSP16 mutants
Studies of CoV MTase mutants have underscored the importance of IFIT family members, especially IFIT1, in mediating attenuation of NSP16-deficient CoVs (Abbas et al., 2017;Menachery et al., 2017Menachery et al., , 2014b;;Russ et al., 2022;Schindewolf et al., 2023;Züst et al., 2011).IFIT family members are highly expressed during IFN-I stimulation, and are also induced by interferon regulatory factor (IRF)− 3 (Grandvaux et al., 2002); therefore, they are an important component of the early antiviral response.IFIT proteins have different affinities for RNA cap structures (Kumar et al., 2014), which can be modulated by their interactions with each other (Johnson et al., 2018) in a species-dependent manner.Human IFIT1 recognizes cap0 structure (Abbas et al., 2017), i.e. an RNA cap lacking 2′-O-methylation, and can homodimerize or bind IFIT2 or IFIT3; it also forms a trimer with both IFIT2 and IFIT3 (Mears and Sweeney, 2018).
Because IFIT1 binds cap0 RNA, it competes with eukaryotic initiation factor (eIF) 4F for binding of RNA cap, impeding 48S ribosomal complex formation and thereby inhibiting translation of cap0 RNA (Kumar et al., 2014).Moreover, IFIT1 was also shown to interact with eIF3 via a yeast two-hybrid screen, and exogenous expression of IFIT1 suppressed translation of a reporter construct in a manner dependent on this interaction (Guo et al., 2000).These findings suggest a model of IFIT1 inhibition of cap0 RNA whereby IFIT1, which associates with eIF3 (Terenzi et al., 2006), out-competes neighboring eIF4F for binding to cap0 RNA, thus restricting translation of cap0 RNA.In addition to its sensitivity to viral cap0 RNA, IFIT1 can also target host cap0 RNAs (Williams et al., 2020).
Interestingly, IFIT proteins have been shown to also interact with components of the IFN-I induction pathway.IFIT1 binds stimulator of interferon genes (STING) to modulate its interactions with other IFN-I pathway components (Li et al., 2009).IFIT3 immunoprecipitated with both MAVS and TANK binding kinase 1 (TBK1) and was found to be necessary for robust IFNβ induction (Liu et al., 2011).It remains unclear whether the cap-binding activities of IFIT proteins are necessary for their interactions with components of IFN-I pathways, or vice versa.Yet, these results indicate that IFIT proteins directly interact with cap0 viral RNA and may also amplify the IFN-I response leading to more antiviral activity.

The role of MDA5 in NSP16 MTase mutant attenuation
Besides IFIT proteins, the cytoplasmic RNA sensor MDA5 also plays a role in recognizing or amplifying the response to CoV RNA cap structures, although the precise mechanism remains unclear.MDA5, a retinoic acid-inducible gene (RIG)-I-like sensor helicase, is a primary sensor of CoVs (Rebendenne et al., 2021;Russ et al., 2022;Sampaio et al., 2021;Yin et al., 2021).It is generally believed to recognize long double stranded RNA (> 2 kb) (Kato et al., 2008), but it has also been implicated in recognizing cap0 RNA which lacks 2′-O-methylation (Russ et al., 2022;Züst et al., 2011).MDA5 subsequently interacts with MAVS to initiate pathways leading to IFN-I induction (Kawai et al., 2005).Using an MHV NSP16 mutant, MDA5 was found to mediate attenuation of viral replication resulting in increased IFN-I induction via IRF-3 activation (Züst et al., 2011).However, MDA5 knockout failed to restore NSP16-mutant MHV replication in vivo.For SARS-CoV NSP16 mutants, loss of MDA5 had no major effect on restoring replication in vitro or in vivo (Menachery et al., 2014a(Menachery et al., , 2014b); yet, MDA5 − /− mice had restored C. Schindewolf and V.D. Menachery lung disease with NSP16-mutant virus infection.Most recently, the increase in IFN-I induction by a SARS-CoV-2 NSP16 mutant was abrogated by nullifying MDA5, although viral replication was not restored to WT levels (Russ et al., 2022).Notably, all CoV NSP16 mutants targeting 2′-O-MTase function remain susceptible to IFN-I pre-treatment prior to infection, suggesting that the role of MDA5 in amplifying the innate immune response may be key to its role in antagonizing NSP16-mutant CoVs.Therefore, MDA5 appears to indirectly antagonize NSP16-mutant CoVs, stimulating induction of IFN-I rather than directly exerting antiviral effects.Additionally, WT CoVs, which are less sensitive to IFN-I than NSP16 mutants, may be able to suppress IFN-I induction by virtue of better viral replication and therefore greater production of IFN-I pathway-suppressing viral proteins that exist in the CoV repertoire (Xia et al., 2020) (Fig. 2).
Using in silico approaches with available structural data (Bobrovs et al., 2021), compounds that bind either the SAM-binding site of NSP16 (Sulimov et al., 2022), the RNA cap-binding site, or both in a bi-substrate mechanism of action (Bobileva et al., 2023;Devkota et al., 2021) have been identified as therapeutic candidates; some candidates have also been evaluated for potential cross-reactivity with host MTases (Bobil ¸eva et al., 2021).Additionally, methods for high-throughput screening of compounds in vitro with activity against SARS-CoV-2 NSP16 have been described (Khalili et al., 2021;Samrat et al., 2023).Building from these screening platforms, several studies have identified compounds with demonstrated activity against NSP16 (Bergant et al., 2022;Bobil ¸eva et al., 2021;Bobrovs et al., 2021;Li et al., 2023a;Omer et al., 2023).
While targeting NSP16 has shown some efficacy, combinations with other therapeutics may offer an even more promising approach.Since NSP16-mutant CoV attenuation is dependent on intact IFN-I pathways and production of IFIT family members (Schindewolf et al., 2023;Züst et al., 2011), synergy between NSP16 inhibitors and IFN-I-targeted therapeutics may be particularly promising.For example, DZNep was shown to synergize with IFNα and remdesivir treatment in vitro for an enhanced antiviral effect (Bergant et al., 2022).Similarly, an additive antiviral effect was observed upon treatment with sinefungin and IFN-I together (Schindewolf et al., 2023).Importantly, synergy between these treatments may be particularly effective in vivo.While antiviral treatments in vivo have been shown to reduce titers within the lung (Kuroda et al., 2023;Pruijssers et al., 2020), recent studies have shown NSP16-mutant SARS-CoV-2 to be more attenuated in nasal wash (Schindewolf et al., 2023), where replication of WT virus is particularly robust (Zhou et al., 2021)

NSP16-Deficiency as a basis for live attenuated vaccines
In addition to presenting an attractive target for antiviral development, NSP16 could also form the basis for a live attenuated vaccine (LAV) platform.NSP16 2′-O-MTase mutants of SARS-CoV and MERS-CoV protected mice from lethal challenge with control virus (Menachery et al., 2017(Menachery et al., , 2014b)).In both studies, serum neutralizing antibody titer induced by vaccination correlated with protection.Recently, a SARS-CoV-2 NSP16 mutant protected hamsters challenged with a clinical SARS-CoV-2 isolate; the NSP16-based LAV afforded sterilizing immunity in both the lung and nasal wash (Ye et al., 2022).Despite this success, however, an attenuated vaccine based on NSP16 mutation alone may still pose a risk in susceptible populations.Prior studies with a SARS-CoV NSP16 mutant showed susceptibility of aged mice to the mutant at high doses and reversion to virulence in immunocompromised models (Menachery et al., 2018).Therefore, single targeting of NSP16 as an attenuated CoV vaccine may be problematic.
While single targeting of NSP16 carries significant risks, properties of the NSP16 mutation allow its use in combination with other attenuating mechanisms.Importantly, NSP16 mutants have no major replication attenuation in permissive cell lines such as Vero (Menachery et al., 2017(Menachery et al., , 2014b;;Schindewolf et al., 2023), allowing for the rescue of combination-mutation attenuated strains.One LAV approach for SARS-CoV targeted NSP16 activity in combination with disruption of the CoV exonuclease, NSP14 (Menachery et al., 2018).The double mutant employed multiple attenuating mutations while delivering protection against heterologous challenge, age-dependent disease, and reversion to virulence in immunocompromised models (Menachery et al., 2018).Additionally, recent LAV studies with porcine epidemic diarrhea virus (PEDV) and infectious bronchitis virus (IBV), two important veterinary CoVs, have utilized combination mutations with NSP16 and other viral proteins (Hou et al., 2019;Keep et al., 2022).Several studies have now characterized attenuated mutants of SARS-CoV-2 that could be used in combination with NSP16 for a LAV (Johnson et al., 2021;Liu et al., 2022;Vu et al., 2022).While the success of the mRNA vaccine platform reduces the likelihood of using LAVs in humans going forward, the combination-attenuation platform could have great utility for CoV pathogens in domestic animals, where other LAVs have been successfully deployed (Brun, 2022).In summary, developing LAVs against CoVs that employ inactivation of multiple viral proteins, including NSP16, to both drive attenuation and prevent reversion to virulence, is a promising strategy.

Exploring other roles of NSP16
Beyond the 2′-O-MTase function of NSP16, NSP16 may still have other roles that warrant additional exploration.Supporting this concept, CoV mutants engineered without NSP16 could not be recovered (Almazán et al., 2006;Schindewolf et al., 2023), and no CoV mutants lacking NSP16 have been reported.One explanation is that NSP16 is a key structural component of the CoV RTC.For example, there is preliminary evidence that NSP16 can form a trimer complex with both NSP10 and NSP14, and that this interaction modulates the exonuclease activity of NSP14 (Matsuda et al., 2022).Furthermore, computational modeling of the SARS-CoV-2 RTC suggests that NSP16 may form an integral component of a large complex involving NSP7, NSP8, NSP10, NSP12, NSP13, NSP14, NSP15, and nucleoprotein (Perry et al., 2021).NSP16 may also play a role in host modulation.SARS-CoV-2 NSP16 was shown to bind U1 and U2 small nuclear RNAs (snRNAs) to disrupt splicing of host mRNAs (Banerjee et al., 2020).This interaction is likely mediated by the RNA-binding site of NSP16, but the physical basis for this interaction remains to be explored, as well as whether 2′-O-MTase function is dispensable for this interaction.Thus, while it may be essential for CoV 2′-O-MTase activity, NSP16 may play other critical roles in viral replication and host translation.Further dissection of these aspects of NSP16 would aid our understanding of CoV infection and possibly lead to additional CoV treatments.

Conclusion
CoV NSP16, via a conserved catalytic tetrad, provides 2′-O-MTase function essential to evasion of host innate immunity.Studies with CoVs harboring a mutation in its catalytic tetrad demonstrated attenuation in vitro and in vivo primarily due to the activity of IFIT family members, which sense a lack of cap-proximal 2′-O-methylation on viral RNA.Antiviral treatments targeting NSP16 function are promising drug candidates and can be used in combinatorial approaches with other therapeutics.Moreover, NSP16 deficiency has shown potential as a part of a live attenuated CoV vaccine platform.In summary, targeting NSP16 disrupts a key aspect of CoV immune evasion and offers a highly conserved enzymatic function that holds promise as a target for mitigating future CoV outbreaks.
. The results suggest increased IFN-I signaling and interferon-stimulated gene (ISG) responses in the nasal passages could possibly enhance NSP16-targeted therapeutics.If true, development of a nasal spray containing an NSP16 2-O-MTase inhibitor could be attractive approach.While additional studies are required, NSP16 2-O-MTase activity represents a highly conserved viral function to target for treatment of both current and future CoVs.

Fig. 2 .
Fig. 2. Model of CoV NSP16 2′-O-Methlytransferase Mutant Attenuation.While CoV double-stranded replication intermediates can be recognized by RIG-I like receptors including MDA5, NSP16-mutant viruses possess RNA lacking 2′-O-methylation (purple "X") on their cap.This RNA lacking 2′O methylation drives attenuation of CoV NSP16 mutants via three, non-mutually exclusive mechanisms: (1) NSP16 mutant RNA is sensed by MDA5 which in turn induces increased type I interferon (IFN)-I production; (2) IFIT1 and other IFIT family proteins directly restricts NSP16 mutant RNA lacking cap 2′-O-methylation; (3) restriction of NSP16 mutant RNA reduces production of CoV IFN-I antagonists augmenting the antiviral state.Each of these mechanisms have been linked to NSP16 mutant attenuation and their contribution may vary across cell, tissue, and species type.